Methods for using riboprimers for strand displacement replication of target sequences

ABSTRACT

Methods, compositions and kits for amplifying a target sequence by strand displacement replication using strand-displacing primers. The method uses primers that have only ribonucleotides or purine ribonucleotides and at least one 2′-substituted pyrimidine-2′-deoxyribonucleotide.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/428,013, filed Nov. 21, 2002. The entire disclosure of whichis specifically incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates generally to methods, compositions andkits for amplifying a target sequence by strand displacementreplication. The method uses strand-displacing primers, having onlyribonucleotides or purine ribonucleotides and at least one2′-substituted pyrimidine-2′-deoxyribonucleotide.

BACKGROUND OF THE INVENTION

Methods for strand displacement amplification of linear templates arewell known in the art. In general, methods for strand displacementamplification of linear templates use some kind of process to digest asequence region at or near the 5′-end of one strand of a double-strandedDNA that has been synthesized using the other strand as a template inorder to provide a complementary region for another primer to anneal tothe template strand. Once annealed to the template, the primer is then“primer extended” by a DNA polymerase that has strand-displacingactivity, thereby displacing the strand in front of the 3′-end of thereplicating DNA. This process, which can be thought of as “liberatingthe primer binding site”, is repeated over and over. Each round ofliberating the primer binding site on the template, annealing of anotherprimer and DNA synthesis results in release or “displacement” of thelast-synthesized DNA strand.

By way of example, but not of limitation, methods for stranddisplacement amplification are disclosed in PCT Patent Publication Nos.WO 02/16639; WO 00/56877; and AU 00/29742 of Takara Shuzo Company; U.S.Pat. Nos. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211;5,733,752; 5,744,311; 5,756,702; and 5,916,779 of Becton Dickinson andCompany; U.S. Pat. Nos. 6,238,868; 6,309,833; and 6,326,173 ofNanogen/Becton Dickinson Partnership; U.S. Pat. Nos. 5,849,547;5,874,260; and 6,218,151 of Bio Merieux; U.S. Pat. Nos. 5,786,183;6,087,133; and 6,214,587 of Gen-Probe, Inc.; U.S. Pat. No. 6,063,604 ofWick et al.; U.S. Pat. No. 6,251,639 and U.S. Patent Application Nos.20010034048; 20030017591; 20030087251; and 20030186234 of Kurn; U.S.Pat. No. 6,410,278; and PCT Publication No. WO 00/28082 of Eiken KagakuKabushiki Kaishi, Tokyo, Japan; U.S. Pat. Nos. 5,591,609; 5,614,389;5,773,733; 5,834,202; and 6,448,017 of Auerbach; and U.S. Pat. Nos.6,124,120; and 6,280,949 of Lizardi, all of which are incorporatedherein by reference.

The methods disclosed in U.S. Pat. Nos. 5,523,204; 5,536,649; 5,624,825;5,631,147; 5,648,211; 5,733,752; 5,744,311; 5,756,702; and 5,916,779 ofBecton Dickinson and Company use a restriction enzyme to liberate theprimer binding site.

The methods disclosed in U.S. Pat. Nos. 5,786,183; 6,087,133; and6,214,587 of Gen-Probe, Inc. use multiple primers, typically with a5′-flap, in the absence of a restriction enzyme to liberate theprimer-binding sites. The methods disclosed in U.S. Pat. No. 6,063,604of Wick et al., use primers designed to have a restriction endonucleasenick site to liberate the primer binding site from the template strand.The methods disclosed by Sagawa et al., in PCT Patent Publication No. WO02/16639 and in PCT Patent Publications Nos. WO 00/56877 and AU 00/29742use a composite primer having a 5′-portion comprisingdeoxyribonucleotides and a 3′-portion comprising ribonucleotides, andthen use RNase H to liberate the primer-binding site at the 5′-end. Themethods disclosed in U.S. Pat. No. 6,251,639 of Kurn use a compositeprimer having a 5′-portion comprising ribonucleotides and a 3′-portioncomprising deoxyribonucleotides, and then use RNase H to liberate theprimer-binding site at the 5′-end of the replicating DNA strand.

While all of these methods result in amplification of single-strandedDNA that is complementary to the template strand, still other methodsand kits are needed that are less expensive and that permit easierdesign of assays for a variety of target sequences.

SUMMARY OF THE INVENTION

The present invention provides a method for amplifying a copy of atarget sequence by repetitive strand-displacing DNA polymerization usinga target sequence comprising a target nucleic acid as a template. Morespecifically, the present invention uses a DNA polymerase that iscapable of strand-displacing DNA synthesis, i.e., wherein, a strand thatis annealed to a template is displaced or released, beginning with its5′-end portion, by the 3′-end of the DNA strand newly synthesized by thepolymerase. The present method enables this repetitive copying of thetemplate strand by use of novel primers and reaction conditions thatpermit, in repetitive succession, strand-displacing DNApolymerase-catalyzed primer extension synthesis of a copy of the targetsequence, then digestion of at least a portion of the 5′-Riboprimerportion of the resulting primer extension product using a low level ofan RNase H enzyme, then annealing of another primer, followed bystrand-displacing primer extension again.

In one aspect the invention provides a method for amplifying a targetnucleic acid sequence including a target nucleic acid by hybridizing aRiboprimer to a single stranded DNA template comprising the targetnucleic acid sequence; optionally hybridizing a blocking oligo to aregion of the template which is 5′ with respect to hybridization of theRiboprimer to the template; extending the Riboprimer with DNApolymerase; and cleaving the annealed Riboprimer with an RNAse H enzymesuch that another Riboprimer hybridizes to the template and repeatsprimer extension by strand displacement, whereby multiple copies of thecomplementary sequence of the target sequence are produced.

In another aspect the invention provides a method of producing amicroarray, including amplifying a target nucleic acid sequence by themethods of the invention; and attaching the amplified products onto asolid substrate to make a microarray of the amplified products.

In another aspect the invention provides a method of producing amicroarray, including amplifying a target nucleic acid sequence by themethods of the invention; and hybridizing the amplified products to amicroarray of nucleic acid molecules immobilized on a surface of a solidphase.

In another aspect the invention provides a composition including acomplex of a template strand; a Riboprimer; and a blocking oligo,wherein the blocking oligo affects cessation of DNA replication of atemplate by DNA polymerase.

In another aspect the invention provides a kit for amplification of atarget nucleic acid sequence, including a Riboprimer and a blockingoligo, wherein the blocking oligo affects cessation of DNA replicationof a template by DNA polymerase.

In another aspect the invention provides a method of generating multiplecopies of a polynucleotide sequence complementary to an RNA sequence ofinterest, by (a) extending a first primer hybridized to a target RNAwith an RNA-dependent DNA polymerase, wherein the first primer is aRiboprimer, whereby a complex comprising a first primer extensionproduct and the target RNA is produced; (b) cleaving RNA in the complexof step (a) with an RNase H enzyme; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a second primer extension product is produced toform a complex of first and second primer extension products; (d)cleaving the Riboprimer in the complex of first and second primerextension products with an RNase H enzyme such that a Riboprimerhybridizes to the second primer extension product; and (e) extending theRiboprimer hybridized to the second primer extension product with aDNA-dependent DNA polymerase; whereby the first primer extension productis displaced, and whereby multiple copies of a polynucleotide sequencecomplementary to the RNA sequence of interest are generated.

Another aspect of the invention is the use of primers in stranddisplacement replication methods of the invention where the primerscomprise only purine ribonucleotides and either only pyrimidineribonucleotides or pyrimidine nucleotides wherein at least onepyrimidine nucleotide includes a pyrimidine 2′-deoxyribonucleotidehaving a non-canonical substituent (i.e., which is not a —H or —OHsubstituent) on the 2′-position of the deoxyribose sugar moiety. Asuitable substituent on the 2′-position of the deoxyribose sugar moietyof a pyrimidine 2′-deoxyribonucleotide is a fluorine substituent.

In other aspects of the invention, the primers maybe both pyrimidine2′-deoxyribonucleotides including a 2′-fluoro-2′-deoxyribonucleotide,conferring resistance to many common RNase A-type ribonucleases (whichspecifically cut RNA at pyrimidine nucleotides).

In still other aspects of the invention, one of the primers may includea pyrimidine nucleotide having a canonical ribonucleotide and the othera pyrimidine nucleotide having a pyrimidine 2′-deoxyribonucleotide witha non-canonical substituent on the 2′-position of the deoxyribose sugarmoiety.

In still other aspects, some or all of one of the pyrimidine nucleotidesin a reaction comprise a 2′-deoxyribonucleotide having an amino, anazido, a methoxy, or another non-canonical substituent on the2′-position of the deoxyribose sugar moiety.

Thus, in contrast to other methods of the art, the strand displacementreplication methods of the present invention use primers comprisingribonucleotides or primers comprising purine ribonucleotides and atleast one 2′-substituted pyrimidine deoxyribonucleotide in order toamplify a target sequence. The primers used in strand displacementmethods and kits of the present invention can be obtained fromcommercial sources at reasonable cost or synthesized inexpensively inhigh yield using a simple and rapid in vitro transcription reaction.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Schematic of a basic embodiment of the invention using aRiboprimer for strand displacement replication of a target sequence.

FIG. 2—Schematic of an embodiment of the invention that uses aRiboprimer having a 5′-portion that is not complementary to the targetsequence as a primer for first-strand primer extension of a targetsequence. Then, synthesis of second-strand DNA takes place by a DNApolymerase or reverse transcriptase under polymerization conditions,whereby the non-complementary portion of the Riboprimer is “copied”. TheRiboprimer therefore has a longer region of complementarity forannealing and the T_(m) of the Riboprimer/second-strand DNA complex ishigher. If the first-strand DNA in this example is complementary to atarget nucleic acid in the sample (e.g., an mRNA target), the stranddisplacement replication reaction shown amplifies the second-strand DNA,thereby synthesizing “anti-sense” DNA.

FIG. 3—Ethidium bromide stained agarose gel of strand displacementreplication products of a ssDNA template. The strand-displacementreplication reaction comprised obtaining Riboprimers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for amplifying a copy of atarget sequence by repetitive strand-displacing DNA polymerization usinga target sequence comprising a target nucleic acid as a template. Morespecifically, the present invention uses a DNA polymerase that iscapable of strand-displacing DNA synthesis, i.e., wherein, a strand thatis annealed to a template is displaced or released, beginning with its5′-end portion, by the 3′-end of the DNA strand newly synthesized by thepolymerase. The present method enables this repetitive copying of thetemplate strand by use of novel primers and reaction conditions thatpermit, in repetitive succession, strand-displacing DNApolymerase-catalyzed primer extension synthesis of a copy of the targetsequence, then digestion of at least a portion of the 5′-Riboprimerportion of the resulting primer extension product using a low level ofan RNase H enzyme, then annealing of another primer, followed bystrand-displacing primer extension again.

Thus, in contrast to the methods for strand displacement amplificationin the art which use a chimeric primer consisting of a 5′-portion or a3′-portion comprising only ribonucleotides or only deoxyribonucleotides,the present invention uses only purine ribonucleotides in both the5′-portion and the 3′-portion. Similarly, the pyrimidine nucleotidesused in the present invention whether they comprise ribonucleotides or a2′-deoxyribonucleotides having a non-canonical 2′-substituent, can be inboth the 5′-portion or the 3′-portion of the primer.

DEFINITIONS Amplifying a Nucleic Acid/AmplificationReaction/Amplification Product

The term “amplifying a nucleic acid” herein means increasing the numberof copies of a target nucleic acid sequence or its complement. Thenucleic acid that is amplified can be DNA or RNA or a mixture of DNA andRNA, including modified DNA and/or RNA. The terms “amplifying RNA” or“amplifying DNA” mean, either, increasing the number of copies of targetnucleic acid having a sequence that is identical to a sequence in thestarting RNA or DNA, or increasing the number of copies of a nucleicacid having a sequence that is complementary to or homologous to asequence in the starting RNA or DNA. The products resulting fromamplification of a nucleic acid molecule or molecules (i.e.,“amplification products”), whether the starting nucleic acid is DNA, RNAor both, can be either DNA or RNA, or a mixture of both DNA and RNAnucleosides or nucleotides, or they can comprise modified DNA or RNAnucleosides or nucleotides. A “copy” does not necessarily mean perfectsequence complementarity or identity to the target sequence. Forexample, copies can include nucleotide analogs such as deoxyinosine ordeoxyuridine, intentional sequence alterations (such as sequencealterations introduced through a primer comprising a sequence that ishybridizable, but not complementary, to the target sequence, and/orsequence errors that occur during amplification).

Template

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase. The synthesized copy is complementary to thetemplate. Both RNA and DNA are always synthesized in the 5′-to-3′direction and the two strands of a nucleic acid duplex always arealigned so that the 5′ ends of the two strands are at opposite ends ofthe duplex (and, by necessity, so then are the 3′ ends). In general, DNApolymerases, including both DNA-dependent (i.e., having a DNA template)and RNA-dependent (i.e., having an RNA template, which enzyme is alsocalled a “reverse transcriptase”) DNA polymerases, require a primer forsynthesis of DNA.

The term “template” is used in different contexts related to the presentinvention. In one context, a target nucleic acid comprising RNA or DNAcan be a “template” for obtaining a ssDNA comprising a target sequenceby primer extension of a suitable DNA primer using a reversetranscriptase or DNA polymerase. In another context, a method of thepresent invention uses a ssDNA comprising the target sequence as a“template” for primer extension of a Riboprimer of the invention using aDNA polymerase. In some embodiments wherein the target nucleic acidcomprises DNA, the ssDNA comprising the target sequence can be useddirectly as an amplification template for primer extension using aRiboprimer of the invention.

The terms “3′-of” and “5′-of” are used herein to refer to the positionof a particular nucleotide, nucleic acid sequence, gene or geneticelement within a nucleic acid or polynucleotide or oligonucleotiderelative to other nucleotides, sequences, genes or genetic elements.Those with knowledge in the art will understand these terms in thecontext of nucleic acid chemistry and structure, particularly related tothe 3′- and 5′-positions of sugar moieties of canonical nucleic acidnucleotides, and in the context of enzymatic synthesis of nucleic acidsin a 5′-to-3′ direction. Those with knowledge in the art will understandthat, if a first nucleic acid sequence is 3′-of a second sequence on onestrand, the complement of the first sequence will be 5′-of thecomplement of the second sequence on the complementary strand.

Primer

A “primer” is an oligonucleotide, generally with a free 3′-OH group,that is complementary to a template and which “binds” (or “complexes” or“anneals” or “hybridizes”), by hydrogen bonding and other molecularforces, to the template to give a primer/template complex for initiationof synthesis by a DNA polymerase, and which is extended (i.e., “primerextended”) by nucleotides being covalently linked its 3′-end with basescomplementary to those at the template in the process of DNA synthesis.The result is a primer extension product. Virtually all DNA polymerases(including reverse transcriptases) that are known require complexing ofan oligonucleotide to a single-stranded template (“priming”) to initiateDNA synthesis, whereas RNA replication and transcription (copying of RNAfrom DNA) generally do not require a primer.

Like templates, primers are used in different contexts related to thepresent invention. The primers are different in each context.

In one context, the primer is used “to prime” primer extension on atemplate comprising a target nucleic acid in order to obtain a ssDNAcomprising a target sequence, but not to prime strand-displacingamplification using a method of the invention. In this context, theprimer usually, but not necessarily, comprises deoxyribonucleotidesrather than ribonucleotides. If a primer comprising ribonucleotides isused in this context, then the ribonucleotides are removed from theresulting ssDNA comprising the target sequence prior to use of the ssDNAas a template for amplification using a Riboprimer in a method of theinvention.

In another context, a Riboprimer is used in methods of the presentinvention in order to prime strand-displacing primer extension using assDNA comprising a target sequence as a template.

Plus/Minus Strand(s)

Discussions of nucleic acid synthesis are simplified and clarified byadopting terms to name the two complementary strands of a nucleic acidduplex. Traditionally, the strand encoding the sequences used to produceproteins or structural RNAs is designated as the “plus” or “sense”strand, and its complement is designated as the “minus” or “anti-sense”strand. It is now known that in many cases, both strands are functional,and the assignment of the designation “plus” to one and “minus” to theother must then be arbitrary. Nevertheless, the terms are useful fordesignating the sequence orientation of nucleic acids or for designatingthe specific mRNA sequences transcribed and/or present in a particularcell, tissue, or sample, and are employed herein for that purpose.

Hybridize/Anneal/Complex/Hybridization/Annealing

The terms “to complex” or “to hybridize” or “to anneal” and “complexing”or “hybridization” or “annealing” refer to the formation of “complexes”or “hybrids” between nucleic acid sequences that are sufficientlycomplementary to bind to each other via Watson-Crick base pairing. Wherea primer “hybridizes” or “anneals” with a template, such complexes (orhybrids) are sufficiently stable to serve the priming function requiredby the DNA polymerase to initiate DNA synthesis.

Nucleic Acids and Polynucleotides of the Invention

A “nucleic acid” or “polynucleotide” of the invention is a polymermolecule comprising a series of “mononucleosides,” also referred to as“nucleosides,” in which the 3′-position of the pentose sugar of onenucleoside is linked by an internucleoside linkage, such as, but notlimited to, a phosphodiester bond, to the 5′-position of the pentosesugar of the next nucleoside. A nucleoside linked to a phosphate groupis referred to as a “nucleotide.” The nucleotide that is linked to the5′-position of the next nucleotide in the series is referred to as “5′of,” or “upstream of,” or the “5′ nucleotide” and the nucleotide that islinked to the 3′-position of the 5′ or upstream nucleotide is referredto as “3′ of,” or “downstream of,” or the “3′ nucleotide.” The pentosesugar of the nucleic acid can be ribose, in which case, the nucleic acidor polynucleotide is referred to as “RNA,” or it can be 2′-deoxyribose,in which case, the nucleic acid or polynucleotide is referred to as“DNA.” Alternatively, especially if the nucleic acid is synthesizedchemically, the nucleic acid can be composed of both DNA and RNAmononucleotides. In both RNA and DNA, each pentose sugar is covalentlylinked to one of four common “nucleic acid bases” (each also referred toas a “base”). Three of the predominant naturally-occurring bases thatare linked to the sugars (adenine, cytidine and guanine) are common forboth DNA and RNA, while one base is different; DNA has the additionalbase thymine, while RNA has the additional base uridine. Those in theart commonly think of a small polynucleotide as an “oligonucleotide.”The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably about 10 to 200 nucleotides, but there is no defined limit tothe length of an oligonucleotide. The exact size will depend on manyfactors, which in turn depends on the ultimate function or use of theoligonucleotide.

Also, for a variety of reasons, a nucleic acid or polynucleotide of theinvention may comprise one or more modified nucleic acid bases, sugarmoieties, or internucleoside linkages. By way of example, some reasonsfor using nucleic acids or polynucleotides that contain modified bases,sugar moieties, or internucleoside linkages include, but are not limitedto: (1) modification of the melting temperature (T_(m)); (2) changingthe susceptibility of the polynucleotide to one or more nucleases; (3)providing a moiety for attachment of a label; (4) providing a label or aquencher for a label; or (5) providing a moiety, such as biotin, forattaching to another molecule which is in solution or bound to asurface.

In order to accomplish the goals of the invention, there is no limit tothe composition of the nucleic acids or polynucleotides of theinvention, including any detection probes, such as, but not limited tomolecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476 of Tyagi etal., and U.S. Pat. No. 6,461,817 of Alland et al., all of which areincorporated herein by reference); capture probes, oligonucleotides, orother nucleic acids used or detected in the assays or methods, so longas each of the nucleic acid functions for its intended use. By way ofexample, but not of limitation, the nucleic acid bases in themononucleotides may comprise guanine, adenine, uracil, thymine, orcytidine, or alternatively, one or more of the nucleic acid bases maycomprise xanthine, allyamino-uracil, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl adenines, 2-propyl and other alkyl adenines,5-halouracil, 5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine,7-deazaadenine, 7-deazaguanine, 7-deaza-7-methyl-adenine,7-deaza-7-methyl-guanine, 7-deaza-7-propynyl-adenine,7-deaza-7-propynyl-guanine and other 7-deaza-7-alkyl or 7-aryl purines,N2-alkyl-guanine, N2-alkyl-2-amino-adenine, purine 6-aza uracil, 6-azacytosine and 6-aza thymine, 5-uracil (pseudo uracil), 4-thiouracil,8-halo adenine, 8-amino-adenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines and 8-halo guanines,8-amino-guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,other aza and deaza thymidines, other aza and deaza cytosine, aza anddeaza adenines, aza and deaza guanines or 5-trifluoromethyl uracil and5-trifluorocytosine. Still further, they may comprise a nucleic acidbase that is derivatized with a biotin moiety, a digoxigenin moiety, afluorescent or chemiluminescent moiety, a quenching moiety or some othermoiety. The invention is not limited to the nucleic acid bases listed;this list is given to show the broad range of bases which may be usedfor a particular purpose in a method.

In some embodiments of the invention, a molecule comprising a “peptidenucleic acid” (PNA) or a molecule comprising both a nucleic acid and aPNA, as described in U.S. Pat. Nos. 5,539,082; 5,641,625; 5,700,922;5,705,333; 5,714,331; 5,719,262; 5,736,336; 5,773,571; 5,786,461;5,817,811; 5,977,296; 5,986,053; 6,015,887; and 6,020,126 (andreferences therein), can also be used. In general, a PNA molecule is anucleic acid analog consisting of a backbone comprising, for example,N-(2-aminoethyl)glycine units, to each of which a nucleic acid base islinked through a suitable linker, such as, but not limited to an aza,amido, ureido, or methylene carbonyl linker. The nucleic acid bases inPNA molecules bind complementary single-stranded DNA or RNA according toWatson-Crick base-pairing rules. However, the T_(m)'s for PNA/DNA orPNA/RNA duplexes or hybrids are higher than the T_(m)'s for DNA/DNA,DNA/RNA, or RNA/RNA duplexes. PNA can provide tighter binding andgreater binding stability than a nucleic acid of similar base sequence(e.g., see U.S. Pat. No. 5,985,563). Also, since PNA is not naturallyoccurring, PNA molecules are highly resistant to protease and nucleaseactivity. PNA can be prepared according to methods know in the art, suchas, but not limited to, methods described in the above-mentionedpatents, and references therein.

When a molecule comprising both a nucleic acid and a peptide nucleicacid (PNA) is used in the invention, modified bases can be used in oneor both parts. For example, binding affinity can be increased by the useof certain modified bases in both the nucleotide subunits that make upthe 2′-deoxyoligonucleotides of the invention and in the peptide nucleicacid subunits. Such modified bases may include 5-propynylpyrimidines,6-azapyrimidines, and N-2, N-6 and O-6 substituted purines including2-aminopropyladenine. Other modified pyrimidine and purine base are alsoexpected to increase the binding affinity of macromolecules to acomplementary strand of nucleic acid.

With respect to nucleic acids or polynucleotides of the invention, oneor more of the sugar moieties can comprise ribose or 2′-deoxyribose, oralternatively, one or more of the sugar moieties can be some other sugarmoiety, such as, but not limited to, 2′-fluoro-2′-deoxyribose, or2′-O-methyl-ribose, or 2′-amino-2′-deoxyribose, or2′-azido-2′-deoxyribose.

The internucleoside linkages of nucleic acids or polynucleotides of theinvention can be phosphodiester linkages, or alternatively, one or moreof the internucleoside linkages can comprise modified linkages, such as,but not limited to, phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkages, which are resistantto some nucleases.

When two different, non-overlapping polynucleotides or oligonucleotideshybridize or anneal to different regions of the same linearcomplementary nucleic acid sequence, and the 3′-end of onepolynucleotide or oligonucleotide points towards the 5′-end of theother, the former may be called the “upstream” polynucleotide oroligonucleotide and the latter the “downstream” polynucleotide oroligonucleotide.

A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3, 5, 10, 15, 20, 25 contiguous nucleotides.

Methods for Making Nucleic Acids and Polynucleotides

A variety of methods are known in the art for making nucleic acids thathave a particular sequence or that contain particular nucleic acidbases, sugars, internucleoside linkages, chemical moieties, and othercompositions and characteristics. Any one or any combination of thesemethods can be used to make a nucleic acid, polynucleotide, oroligonucleotide for the present invention. These methods include, butare not limited to:

(1) chemical synthesis (usually, but not always, using a nucleic acidsynthesizer instrument);

(2) post-synthesis chemical modification or derivatization;

(3) cloning of a naturally occurring or synthetic nucleic acid in anucleic acid cloning vector (e.g., see Sambrook, et al., MolecularCloning: A Laboratory Approach 2^(nd) ed., Cold Spring Harbor LaboratoryPress, 1989) such as, but not limited to a plasmid, bacteriophage (e.g.,m13 or lamba), phagemid, cosmid, fosmid, YAC, or BAC cloning vector,including vectors for producing single-stranded DNA;

(4) primer extension using an enzyme with DNA template-dependent DNApolymerase activity, such as, but not limited to, Klenow, T4, T7, rBst,Taq, Tfl, Tth or phi29 DNA polymerases (U.S. Pat. Nos. 5,576,204 and5,001,050 to Blanco et al., incorporated herein by reference; phi29 isavailable under the trademark name RepliPHI™ from EpicentreTechnologies, Madison, Wis., USA), including mutated, truncated (e.g.,exo-minus), or chemically-modified forms of such enzymes;

(5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds., PCR Primer: ALaboratory Manual, 1995, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.);

(6) reverse transcription (including both isothermal synthesis andRT-PCR) using an enzyme with reverse transcriptase activity, such as,but not limited to, reverse transcriptases derived from avianmyeloblasosis virus (AMV), Maloney murine leukemia virus (MMLV),Bacillus stearothermophilus (rBst), or Thermus thermophilus (Tth);

(7) in vitro transcription using an enzyme with RNA polymerase activity,such as, but not limited to, SP6, T3, or T7 RNA polymerase, Tth RNApolymerase, E. coli RNA polymerase, or SP6 or T7 R&DNA™ Polymerase(EPICENTRE Technologies, Madison, Wis., USA), or another enzyme;

(8) use of restriction enzymes and/or modifying enzymes, including, butnot limited to exo- or endonucleases, kinases, ligases, phosphatases,methylases, glycosylases, terminal transferases, including kitscontaining such modifying enzymes and other reagents for makingparticular modifications in nucleic acids;

(9) use of polynucleotide phosphorylases to make new randomized nucleicacids;

(10) other compositions, such as, but not limited to, a ribozyme ligaseto join RNA molecules; and/or

(11) any combination of any of the above or other techniques known inthe art. Oligonucleotides and polynucleotides, including chimeric (i.e.,composite) molecules and oligonucleotides with modified bases, sugars,or internucleoside linkages are commercially available (e.g., fromTriLink Biotechnologies, San Diego, Calif., USA or Integrated DNATechnologies, Coralville, Iowa).

Target Nucleic Acids and Target Nucleic Acid Sequences

A “target nucleic acid” has a “target sequence” to be amplified, and maybe either single-stranded or double-stranded and may include othersequences besides the target sequence which may not be amplified. Atarget nucleic acid is sometimes referred to more specifically by thetype of nucleic acid. By way of example, but not of limitation, a targetnucleic acid can be a “target RNA” or an “RNA target,” or a “targetmRNA,” or a “target DNA” or a “DNA target.” Similarly, the targetsequence can be referred to as “a target RNA sequence” or a “RNA targetsequence”, or as a “target mRNA sequence” or a “target DNA sequence,” orthe like. In some embodiments, the target sequence comprises one or moreentire target nucleic acids, such as, but not limited to, mRNA targetnucleic acids in a particular cell. In other embodiments, the targetsequence comprises only a portion of one or more nucleic acid molecules.The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid(s) that is/are to be amplified. The “targetsequence” includes the complexing sequences to which theoligonucleotides (primers and/or splice templates) complex during theprocesses of the present invention, including “tail” sequences which areadded by means including, but not limited to, non-templated addition ofdCMP residues to first-strand cDNA by reverse transcriptase pausing atcap structures of mRNA (in the presence or absence of manganese cations)and/or controlled ribonucleotide tailing using TdT. When the targetnucleic acid is originally single-stranded, the term “target sequence”is also meant to refer to the sequence complementary to the “targetsequence.” When the “target nucleic acid” is originally double-stranded,the term “target sequence” refers to both the (+) and (−) strands. Thetarget sequence may be known or not known, in terms of its actualsequence. In some instances, the terms “target sequence,” “targetnucleic acid,” “target polynucleotide,” and variations thereof, are usedinterchangeably.

A target nucleic acid, comprising a target sequence to be amplified,includes nucleic acids from any biological source, whether living ordead, in purified or unpurified form. Target nucleic acids can be anysingle-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), including,but not limited to mitochondrial DNA, chloroplast DNA, chromosomes,plasmids or other episomes, the genomes of bacteria, yeasts, viruses,viroids, mycoplasma, molds, or other microorganisms, or genomes offungi, plants, animals, or humans, or target nucleic acids can be anyRNA, including, but not limited to tRNA, mRNA, rRNA, mitochondrial RNA,chloroplast RNA, or target nucleic acids can be mixtures of DNA and RNA,including, but not limited to, mixtures of the above nucleic acids orfragments thereof, or DNA-RNA hybrids. The target nucleic acid can beonly a minor fraction of a complex mixture such as a biological sampleand can be obtained from various biological materials by proceduresknown in the art. Methods for purification of a target nucleic, iffurther purification is necessary, are also known in the art that can beused to obtain a target nucleic acid for use in a method of theinvention.

A. General Methods for Obtaining a Target Sequence

An initial step in obtaining a target nucleic acid sequence is renderingthe target nucleic acid single-stranded. If the target nucleic acid is adsDNA, the initial step is target denaturation. The denaturation stepmay be thermal denaturation or any other method known in the art, suchas alkali treatment.

In some embodiments of the invention in which the target nucleic acid ina sample is DNA, the ssDNA target sequence comprises either ssDNA thatis present in a biological sample or ssDNA that is obtained bydenaturation of dsDNA in the sample.

In other embodiments, the ssDNA target sequence comprises ssDNA that isobtained as a result of a “primer extension reaction,” meaning an invitro or in vivo DNA polymerization reaction using either ssDNA ordenatured dsDNA that is present in the sample as a template and anoligonucleotide as a primer under DNA polymerization reactionconditions.

In some embodiments the target nucleic acid in the sample or the primerextension product, or both, are made into smaller DNA fragments bymethods known in the art in order to generate a DNA target sequence foruse in the methods of the invention.

In some embodiments using samples containing DNA target nucleic acids, assDNA target sequence is obtained by a strand displacement reactionusing a strand displacement method of the present invention. In otherembodiments, a ssDNA target sequence is obtained using another stranddisplacement method, such as but not limited to the methods described inPCT Patent Publication Nos. WO 02/16639; WO 00/56877; and AU 00/29742 ofTakara Shuzo Company, Kyoto, Japan; U.S. Pat. Nos. 5,523,204; 5,536,649;5,624,825; 5,631,147; 5,648,211; 5,733,752; 5,744,311; 5,756,702; and5,916,779 of Becton Dickinson and Company; U.S. Pat. Nos. 6,238,868;6,309,833; and 6,326,173 of Nanogen/Becton Dickinson Partnership; U.S.Pat. Nos. 5,849,547; 5,874,260; and 6,218,151 of Bio Merieux; U.S. Pat.Nos. 5,786,183; 6,087,133; and 6,214,587 of Gen-Probe, Inc.; U.S. Pat.No. 6,063,604 of Wick et al.; U.S. Pat. No. 6,251,639 of Kurn; U.S. Pat.No. 6,410,278; and PCT Publication No. WO 00/28082 of Eiken KagakuKabushiki Kaishi, Tokyo, Japan; U.S. Pat. Nos. 5,591,609; 5,614,389;5,773,733; 5,834,202; and 6,448,017 of Auerbach; and U.S. Pat. Nos.6,124,120; and 6,280,949 of Lizardi, all of which are incorporatedherein by reference. In still other embodiments the ssDNA targetsequence is obtained from a rolling circle replication reaction. The3′-end of the DNA target sequence can be defined, if it need be defined,by using any suitable method known in the art, such as, but not limitedto a method discussed herein below.

If the target nucleic acid is RNA, the initial step for obtaining atarget sequence can be the synthesis of a single-stranded cDNA. Ingeneral, “cDNA” refers herein to “complementary DNA” that is synthesizedby primer extension by a DNA polymerase, including, but not limited to,an RNA-dependent DNA polymerase, using at least a portion of a nucleicacid as a template, and which cDNA is “homologous to” or “base pairswith” or “complexes with” at least a portion of the nucleic acidtemplate. In some embodiments of the invention, a target sequencecomprises cDNA that is synthesized by reverse transcription primerextension by an RNA-dependent DNA polymerase (i.e., “reversetranscriptase”) using a target nucleic acid comprising messenger RNA(mRNA) obtained from a biological sample as a template, which cDNA ishomologous to the mRNA. In other embodiments, a target sequencecomprising cDNA is obtained by reverse transcriptase primer extensionusing an RNA target that is not mRNA as a template, or by primerextension using a single-stranded DNA target or one strand of adouble-stranded DNA target as a template.

Techniques for the synthesis of cDNA from RNA are known in the art asdescribed herein. Thus, in some embodiments of the invention, the targetnucleic acid is RNA and the ssDNA target sequence comprises first-strandcDNA obtained by reverse transcription of the RNA target, meaning an invitro reaction that utilizes an RNA present in a sample as a templateand a nucleic acid oligonucleotide that is complementary to at least aportion of a sequence of the RNA template as a primer in order tosynthesize ssDNA using an RNA-dependent DNA polymerase (i.e., reversetranscriptase) under reaction conditions.

An “RNA-dependent DNA polymerase” or “reverse transcriptase” is anenzyme that synthesizes a complementary DNA copy (“cDNA”) from an RNAtemplate. All known reverse transcriptases also have the ability to makea complementary DNA copy from a DNA template; thus, they are both RNA-and DNA-dependent DNA polymerases. A primer is required to initiatesynthesis with both RNA and DNA templates. Examples of reversetranscriptases that can be used in methods of the present inventioninclude, but are not limited to, AMV reverse transcriptase, MMLV reversetranscriptase, Tth DNA polymerase, rBst DNA polymerase large fragment,also called IsoTherm™ DNA Polymerase (EPICENTRE Technologies, Madison,Wis., USA), and BcaBEST™ DNA polymerase (Takara Shuzo Co, Kyoto, Japan).In some cases, a mutant form of a reverse transcriptase, such as, butnot limited to, an AMV or MMLV reverse transcriptase that lacks RNase Hactivity is suitable.

In some embodiments, a first-strand cDNA for use in methods of theinvention is synthesized in situ in cells or tissue in a tissue sectionusing methods similar to those described in U.S. Pat. Nos. 5,168,038;5,021,335; and 5,514,545, which are incorporated herein by reference.Thus, the first-strand cDNA is synthesized by contacting the cells ortissue in the tissue section under hybridizing conditions with a primer,wherein the primer hybridizes to one or more target sequences in thecell or tissue, and then contacting the primer-mRNA complex with areverse transcriptase under reverse transcription reaction conditions.

An oligonucleotide primer for synthesis of cDNA using an RNA target as atemplate can be complementary to a specific known sequence in the RNAtarget in a sample, or the oligonucleotide primer(s) can comprise amixture of all possible or many possible sequences, such as, but notlimited to, random hexamer primers. Random primers can be made byincluding nucleotide reagents that are complementary to each of the fourcanonical bases (i.e., all four nucleotides) during the chemicalsynthesis of each nucleotide position of the oligonucleotide that iscomplementary to the target sequence. In embodiments of the inventionusing samples containing mRNA targets, the ssDNA target sequencecomprises first-strand cDNA that is made by reverse transcription of themRNA using an oligonucleotide primer comprising either a specificsequence which is complementary to a known sequence of a specific mRNAor, if the mRNA has a poly(A) tail at its 3′-end, an oligo(dU) primer oran oligo(dU) anchor primer.

B. Methods for Defining the 5′- and 3′-Ends of Target Sequences thatComprise Only a Portion of a Larger RNA or DNA Target Nucleic Acid

When a method of the invention is used to amplify the completesequence(s) of one or a multitude of nucleic acid molecules, such as,but not limited to, the complete sequences of mRNA molecules (excludingthe cap structure) in a sample, it is not necessary to devise additionalmethods to define the 5′- and 3′-ends of the sequences. However, if amethod of the invention is used to amplify a target sequence thatcomprises only a portion of a larger RNA or DNA nucleic acid in asample, then additional methods are needed to delimit the targetsequence that comprises the template sequence that is amplified.

There are two general approaches to delimiting the ends of the targetsequence that is amplified. In the first direct approach, methods areused to determine the size and end sequences of a target nucleic acidmolecule or molecules present in the sample itself. In the secondindirect approach, instead of changing the size and end sequences of thetarget nucleic acid molecules present in a sample, methods are used todetermine the size and end sequences of one or more first-strand cDNAmolecules that is synthesized by reverse transcription or primerextension, respectively, of RNA or of at least one strand of DNA in asample.

With respect to the direct approach, a number of methods are known inthe art for cleaving a nucleic acid molecule at or near a specificsequence, and any of the methods which delimit the size and endsequences of a target nucleic acid for an application of the presentinvention can be used. By way of example, but not of limitation, a DNAin a sample comprising a dsDNA molecule or a ssDNA molecule to which anappropriate complementary DNA oligo is annealed can be digested with arestriction endonuclease, provided a restriction site that would providea suitable 5′-end and/or 3′-end sequence is present. Alternatively, oneor more DNA oligonucleotides having a double-stranded segment thatcontains a FokI restriction enzyme site and a single-stranded segmentthat binds to the desired cleavage site on a first-strand cDNA can beused. As is well known in the art, this type of oligonucleotide can beused with the restriction enzyme FokI to cut a single-stranded DNA atalmost any desired sequence (Szybalski, W., Gene, 40: 169-173, 1985;Podhajska A. J. and Szybalski W., Gene 40:175, 1985, incorporated hereinby reference).

By way of further example, but not of limitation, a ssRNA target nucleicacid present in a sample can be cleaved using a ribonuclease H inregions to which complementary oligonucleotides comprising at leastthree-to-four deoxyribonucleotides, and preferably four to tendeoxyribonucleotides, are annealed. Alternatively, a linear DNAoligonucleotide can be annealed to an RNA in a sample at a location thatencodes a recognition site of a restriction enzyme that can cut RNA:DNAheteroduplexes. Cutting the target RNA:DNA oligo with the enzyme willthen generate a defined end. Alternatively, an RNA or DNA oligo orpolynucleotide with a sequence complementary to the region of an RNAtarget sequence that is intended to become a substrate for amplificationcan be annealed to the RNA and the sequences of the RNA to which theoligo or polynucleotide is not annealed can be digested using asingle-strand-specific ribonuclease, such as RNase A or RNase T1. Stillfurther, either RNA or DNA nucleic acids of known sequence can becleaved at specific sites using a 5′-nuclease or Cleavase® enzyme andspecific oligonucleotides, as described by Kwiatkowski, et al.,(Molecular Diagnosis, 4: 353-364, 1999) and in U.S. Pat. No. 6,001,567and related patents assigned to Third Wave Technologies (Madison, Wis.,USA), which are incorporated herein by reference.

In general, with respect to the second indirect approach, the 5′-end ofthe primer that is used for reverse transcription of RNA in a sample orfor primer extension of at least one strand of DNA in a sample definesthe 5′-end of the first-strand cDNA target sequence that is amplified inthe methods of the present invention. Thus, a sample target nucleic acidthat is reverse transcribed or primer extended to make a first-strandcDNA target sequence need not have a defined 3′-end.

In order to generate a defined 3′-end on a first-strand cDNA (i.e.,corresponding to the 5′-end of the target sequence), a number of methodsknown in the art may be used, all of which are envisioned as methods ofthe present invention. By way of example, but not of limitation, if aspecific sequence is present in the first-strand cDNA that correspondsto a restriction endonuclease site that would provide a suitable 3′-endsequence, a complementary DNA oligo can be annealed to this sequence andthe site can be cleaved with the restriction enzyme. The DNA oligo mayoptionally have a 2′,3′-dideoxy end, a 2′-amino end, or another end sothat it cannot be extended by a DNA polymerase. Alternatively, the3′-end can be defined using a DNA oligonucleotide having adouble-stranded segment that contains a FokI restriction enzyme site anda single-stranded segment that binds to the desired cleavage site on afirst-strand cDNA (Szybalski, W., Gene, 40: 169-173, 1985; Podhajska A.J. and Szybalski W., Gene 40:175, 1985), as discussed previously. Stillfurther, a 5′-nuclease can be used to cleave a first-strand cDNA at adefined 3′-end as discussed above.

In addition to the above methods, the 3′-end of a first-strand cDNA canalso be limited by other methods. A preferred method of the invention isto use a “blocking oligo” or a “blocker sequence,” as disclosed byLaney, et al., in U.S. Pat. No. 5,679,512, and by Kurn in U.S. Pat. No.6,251,639, both of which are incorporated herein by reference. The“blocker sequence” or “blocker oligo” is a polynucleotide, which isusually a synthetic polynucleotide that is single-stranded and comprisesa sequence that is hybridizable, and preferably complementary, to asegment of target nucleic acid, wherein the blocking oligo anneals tothe target nucleic acid so as to block further primer extension of the3′-end of first-strand cDNA at a desired position. Some embodiments ofthe processes of the strand displacement replication methods of thepresent invention use a blocking oligo. The blocking oligo comprisesnucleotides that bind to the target nucleic acid with an affinity,preferably a high affinity, such that the blocker sequence resistsdisplacement by DNA polymerase in the course of primer extension, inpreferably more than about 30%, more preferably more than about 50%,even more preferably more than about 75%, and most preferably more thanabout 90%, of primer extension events. The length and composition of theblocker polynucleotide should be such that excessive random non-specifichybridization is avoided under the conditions of the methods of thepresent invention. The length of the blocker polynucleotide ispreferably from about 3 to about 30 nucleotides, more preferably fromabout 5 to about 25 nucleotides, even more preferably from about 8 toabout 20 nucleotides, and most preferably from about 10 to about 15nucleotides. In other embodiments, the blocker polynucleotide is atleast about any of the following: 3, 5, 8, 10, 15; and less than aboutany of the following: 20, 25, 30, 35. It is understood that the lengthcan be greater or less as appropriate under the reaction conditions ofthe methods of this invention. The complementarity of the blockerpolynucleotide is preferably at least about 25%, more preferably atleast about 50%, even more preferably at least about 75%, and mostpreferably at least about 90%, to its intended binding sequence on thetarget nucleic acid. In some embodiments, the blocker sequence thathybridizes to a DNA target nucleic acid is attached to the DNA such thatdisplacement of the blocker sequence by the polymerase that effectsprimer extension is substantially, or at least sufficiently, inhibited.Suitable methods for achieving such attachment include techniques knownin the art, such as using a cytosine analog that contains a G-clampheterocycle modification as described by Flanagan et al., (Proc. Natl.Acad. Sci. USA 96: 3513-3518, 1999); and locked nucleic acids asdescribed, e.g., by Kumar et al., (Bioorg. Med. Chem. Lett., 8:2219-2222, 1998; and by Wahlestedt et al., (Proc. Natl. Acad. Sci. USA97: 5633-5638, 2000), all of which are incorporated herein by reference.Other suitable methods include using, where appropriate, sequences witha high GC content and/or cross-linking. Any of these methods forobtaining enhanced attachment may be used alone or in combination.Alternatively, a molecule comprising a peptide nucleic acid (PNA) can beused.

Still further, another method that can be used to limit the 3′-end of afirst-strand cDNA is to use a thermocycler with short DNA synthesiselongation cycles during reverse transcription or primer extension tosynthesize first-strand cDNA. The length of the product can be somewhatcontrolled by the length of the DNA synthesis cycle. Conditions can bedetermined to define an approximate chain length of first-strand cDNA bycontrolling the temperature and time interval of DNA synthesis beforedenaturing the growing first-strand cDNA from the template by raisingthe temperature.

Further, the 3′-end of a first-strand cDNA that is to become thetemplate sequence for a strand displacement replication reaction can bedefined by first amplifying the target nucleic acid sequence usinganother amplification method that delimits the end sequence. By way ofexample, but not of limitation, it can be prepared using PCR, RT-PCR,NASBA, TMA, 3SR, Ligation Chain Reaction (LCR), Linked LinearAmplification (BioRad), SDA, RCA, ICAN™ (Takara: Sagawa et al., in PCTPatent Publication No. WO 02/16639; and in PCT Patent Publications Nos.WO 00/56877 and AU 00/29742); or a strand-displacement method of Kurn(U.S. Pat. No. 6,251,639), all of which are incorporated herein byreference.

If a 3′-end of a target sequence need not be at an exact location, andcan be random or imprecise, which is the case in some embodiments of theinvention, there are a number of other methods that can be used formaking smaller fragments of a DNA molecule, whether for a target nucleicacid, a target sequence, or otherwise. By way of example, but not oflimitation, a target nucleic acid can be fragmented by physical means,such as by movement in and out of a syringe needle or other orifice orby sonication, preferably with subsequent end repair, such as using a T4DNA polymerase or a kit, such as the End-It™ DNA End Repair Kit(EPICENTRE Technologies, Madison, Wis., USA).

Still another method that can be used is to incorporate dUMP randomlyinto the first-strand cDNA during reverse transcription or primerextension by using dUTP in place of a portion of the TTP in thereaction. The dUMP will be incorporated randomly in place of TMP at afrequency based on the ratio of dUTP to TTP. Then, the first-strand cDNAcan be cleaved at sites of dUMP incorporation by treatment (e.g., seeU.S. Pat. No. 6,048,696, incorporated herein by reference) withuracil-N-glycosylase (UNG) and endonuclease IV (endo IV), which areavailable from EPICENTRE Technologies (Madison, Wis., USA). UNGhydrolyzes the N-glycosidic bond between the deoxyribose sugar anduracil in single- and double-stranded DNA that contains uracil in placeof thymidine. It has no activity on uracil residues in RNA or on dUTP.Endo IV cleaves the phosphodiester linkage at the basic site. It may beuseful to use a thermolabile UNG (e.g., HK™-UNG from EPICENTRETechnologies, Madison, Wis., USA) for some applications. (Also,incorporation of dUMP at specific sites within a syntheticoligonucleotide or, for example, within a promoter primer of theinvention between the 3′-target-sequence-complementary portion and thepromoter sequence, introduces specific cleavage sites which can be usedat any time to cleave a resulting nucleic acid which contains the siteby treatment with UNG and endo IV.)

Still further, in some cases, the 3′-end of a first-strand cDNA can bedefined by treatment with exonuclease III (Henikoff, S., Gene, 28: 351,1984). In still other cases, the 3′-end of a first-strand cDNA that isannealed to a DNA target nucleic acid can be incubated with T4 DNApolymerase or unmodified T7 DNA polymerase in the absence or thepresence of dNTPs in the reaction (2002 EPICENTRE Catalog, pp. 129 and130); these enzymes have the 3′-to-5′ exonuclease activity in theabsence of dNTPs, but the polymerase activity predominates in thepresence of dNTPs. These are only some of the methods that can be usedto define the 3′-ends of a first-strand cDNA, and the invention is notlimited to these methods, which are presented only as examples.

General Methods of the Invention

The following are general methods of the invention which are furthercharacterized and supported by the detailed descriptions hereinbelow.

In one embodiment, the invention provides a method for amplifying atarget nucleic acid sequence comprising a target nucleic acid, themethod comprising:

(a) hybridizing a Riboprimer to a single-stranded DNA templatecomprising the target nucleic acid sequence;

(b) optionally, hybridizing a blocking oligo to a region of the templatewhich is 5′ with respect to hybridization of the Riboprimer to thetemplate;

(c) extending the Riboprimer with DNA polymerase; and

(d) cleaving the annealed Riboprimer with an enzyme that cleaves RNAfrom an RNA/DNA hybrid such that another Riboprimer hybridizes to thetemplate and repeats primer extension by strand displacement, wherebymultiple copies of the complementary sequence of the target sequence areproduced.

In another embodiment, the invention provides for a method foramplifying a target nucleic acid sequence comprising a target nucleicacid, the method comprising:

(a) obtaining a single-stranded DNA comprising a target nucleic acidsequence;

(b) obtaining a Riboprimer, the Riboprimer comprising ribonucleotides,wherein at least the 3′-end portion of the Riboprimers is complementaryto the 3′-end portion of the target nucleic acid sequence;

(c) optionally, obtaining a blocking oligo and annealing the blockingoligo to a region of the single-stranded DNA, wherein the 5′-end of theblocking oligo that is annealed to the single-stranded DNA delimits the3′-end of the target nucleic;

(d) annealing the Riboprimer and the blocking oligo, if a blocking oligois used, to the single-stranded DNA;

(e) obtaining a strand-displacing DNA polymerase;

(f) primer extending the Riboprimer annealed to the single-stranded DNAwith the strand-displacing DNA polymerase under strand-displacingpolymerization conditions;

(g) obtaining a double-stranded complex comprising the single-strandedDNA and a primer extension product, wherein the primer extension productcomprises the Riboprimer sequence in its 5′-end portion and the targetsequence in its 3′-end portion;

(h) contacting the double-stranded complex with an RNase H enzyme underenzyme reaction conditions so as to release at least a portion of theRiboprimer sequence in the 5′-end portion of the primer extensionproduct of the double-stranded complex;

(i) annealing a second Riboprimer, which is identical to the Riboprimerof step (b), to the single-stranded DNA of the double-stranded complex,wherein the second Riboprimer anneals to the single-stranded DNA at theposition where the portion of the Riboprimer sequence of the primerextension product was released;

(j) primer extending the second Riboprimer annealed to thesingle-stranded DNA of the double-stranded complex with thestrand-displacing DNA polymerase under strand-displacing polymerizationconditions, so as to displace the first primer extension product fromthe double-stranded complex and obtain a second double-stranded complexcomprising the single-stranded DNA and a second primer extensionproduct;

(k) obtaining the primer extension product that was displaced from thedouble-stranded complex as a result of extending the second Riboprimerannealed to the single-stranded DNA;

(l) repeating steps b through l whereby multiple copies of the primerextension products corresponding to the target sequence are produced;and

(m) optionally, detecting or quantifying the primer extension productsproduced and released from the double-stranded complex.

In yet another embodiment, the invention provides for a method ofamplifying a target nucleic acid sequence complementary to a targetnucleic acid, the method comprising:

(a) hybridizing a primer, the primer comprising a polynucleotidecomprising canonical purine ribonucleotides and non-canonical2′-fluoro-pyrimidine nucleotides, to a target nucleic acid, the targetnucleic acid comprising single-stranded DNA comprising the targetnucleic acid sequence;

(b) optionally, hybridizing a blocking oligo to a region of the targetnucleic acid, wherein the 5′-end of the blocking oligo that is annealedto the target nucleic acid delimits the 3′-end of the extension productthat is to be synthesized using the primer;

(c) extending the primer with DNA polymerase; and

(d) cleaving the primer with an RNase H enzyme so as to liberate atleast a portion of the primer-binding site on the target nucleic acid towhich the primer is complementary, such that another primer hybridizesto the primer binding site on the target nucleic acid and repeats primerextension by strand displacement, whereby multiple copies of thecomplementary sequence to the target sequence are produced.

Strand-displacement methods of the present invention can be combinedwith use of other methods in the art, such as, but not limited tomethods that add a promoter or protopromoter sequence for an RNApolymerase, such as an N4 mini-vRNAP or T7 RNAP or another T7-type RNAP,in order to obtain additional transcription-based amplification of atarget sequence. These methods are further characterized and supportedby the detailed descriptions hereinbelow.

Riboprimers of the Invention

A Riboprimer (or Riboprimer oligo) of the invention is defined herein asan oligonucleotide comprised of four or more ribonucleotides, preferablyabout 10 to 200 ribonucleotides, and most preferably about 20 to 50ribonucleotides, but there is no defined limit to the length of aRiboprimer. The exact size will depend on many factors, including thetype and length of the target nucleic acid and the type and length ofthe target sequence that is replicated using the method. The exact sizeof the Riboprimer will also depend on the mode of performance of themethod, including whether it is performed in a stepwise fashion or in acontinuous fashion, with all steps occurring in the same reactionmixture without further addition of reagents or reaction substrates orcomponents. If a target sequence is long and the purpose for which themethod is used does not require a multiplicity of products of similarsize which span approximately the entire length of the target sequence,a method of the invention can also use multiple Riboprimers to amplify asingle target sequence in various portions, which could be used, forexample, as a multiplicity of probes for a particular target sequence.Multiple Riboprimers can also be used in a single reaction mixture toamplify multiple different non-contiguous target sequences in a sample,which can be for the same or different target nucleic acids.

At least a portion of a Riboprimer of the invention comprises a sequencethat is complementary to the 3′-end portion of a target sequence that isdesired to be amplified, which target-complementary sequence comprisesat least the 3′-end portion of the Riboprimer. The target-complementarysequence at the 3′-end portion of a Riboprimer can be a sequence that iscomplementary to one specific target sequence or thetarget-complementary portion of a Riboprimer can comprise anoligo(dU)_(n) sequence, or an anchored oligo(dU)_(n)X sequence or arandomized sequence in order to amplify a multiplicity of targetsequences, such as, but not limited to target sequences comprising allmRNA targets in a sample. The 3′-end portion that is complementary tothe target sequence must be of sufficient length and have a composition(i.e., T_(m)) so that the 3′-end portion of the Riboprimer anneals tothe target sequence under the annealing conditions of the reaction so asto permit primer extension by the strand-displacing DNA polymerase used.

The entire sequence of the Riboprimer can be complementary to the 3′-endportion of the target sequence, or only a portion of the Riboprimer,i.e., the 3′-end portion, can be complementary to the target sequence,in which case, the 5′-end portion of the Riboprimer need not becomplementary to the target sequence. The target sequence that isamplified using a method of the invention can be identical to a targetsequence in the target nucleic acid in a sample or it can be a sequencethat is complementary to that of the target nucleic acid in the sample.In other words, the template strand for a strand-displacementreplication reaction of the invention can be first-strand cDNA orsecond-strand cDNA.

One reason to use a Riboprimer with a 5′-portion that is notcomplementary to the target sequence is to provide a sequence that canbe copied by second-strand primer extension using as a template afirst-strand cDNA primer extension product made using the Riboprimer.Thus, the resulting second-strand cDNA will have a 3′-end portioncomprising a sequence that is complementary to the 5′-portion of theRiboprimer which is not complementary to the target sequence. Theadditional sequences that are complementary to the 5′-portion of theRiboprimer provide an additional primer-binding site, which can provideimproved amplification for some target sequences, especially if thetarget sequence comprises a multiplicity of target nucleic acidmolecules, such as, for example, a mixture of all polyadenylated mRNAtargets in a sample. Thus, some embodiments of the invention compriseuse of a Riboprimer to obtain a second-strand cDNA template forstrand-displacement replication, wherein the product of the reactioncomprises anti-sense cDNA.

With respect to chemical composition, a Riboprimer of the presentinvention comprises only purine ribonucleotides and either onlypyrimidine ribonucleotides or pyrimidine nucleotides wherein at leastone of the pyrimidine nucleotide comprises a pyrimidine2′-deoxyribonucleotide having a non-canonical substituent (i.e., whichis not a —H or —OH substituent) on the 2′-position of the deoxyribosesugar moiety. A preferred substituent on the 2′-position of thedeoxyribose sugar moiety of a pyrimidine 2′-deoxyribonucleotide is afluorine substituent. In some embodiments of the invention, bothpyrimidine 2′-deoxyribonucleotides comprise a2′-fluoro-2′-deoxyribonucleotide, which confers resistance to manycommon A-type ribonucleases (which specifically cut RNA at pyrimidinenucleotides), whereas in other embodiments, one of the pyrimidinenucleotides comprises a canonical ribonucleotide and the otherpyrimidine nucleotide comprises a pyrimidine 2′-deoxyribonucleotidehaving a non-canonical substituent on the 2′-position of the deoxyribosesugar moiety, such as, but not limited to a 2′-fluorine substituent. ARiboprimer that has purine ribonucleotides and pyrimidine2′-deoxyribonucleotides having a 2′-fluorine substituent can still bedigested using an RNase H, such as Hybridase™ thermostable RNase, whichis important to permit release of the Riboprimer portion of theprimer-extended Riboprimer after DNA synthesis.

In still other embodiments, some or all of one of the pyrimidinenucleotides in a reaction comprises a 2′-deoxyribonucleotide having anamino, an azido, a methoxy, or another non-canonical substituent on the2′-position of the deoxyribose sugar moiety. The sugar moieties havingan amino or azido substituent provide a site for labeling with afluorescent or other chemical moiety, such as, but not limited to a Cy(Cyanine) dye, that permits detection of the strand-displacementproducts when they are used as a probe (i.e., for gene expressionscreening using an array or microarray). Many chemicals and methods forlabeling a probe are well known in art. The invention is not limitedwith respect to the method for labeling a probe. It is envisioned thatany chemical or method that results in a detectable signal for anintended purpose can be used. Riboprimers can be synthesized using anoligonucleotide synthesizer using methods known in the art or they canbe purchased from commercial sources such as TriLink Biotechnologies(San Diego, Calif.) or Integrated DNA Technologies (Coralville, Iowa).Alternatively, Riboprimers can be synthesized in high yields by in vitrotranscription. Riboprimers comprising only ribonucleotides can besynthesized using the AmpliScribe™ T7-Flash™ Transcription Kit accordingto the instructions of the manufacturer (EPICENTRE Technologies,Madison, Wis.). Riboprimers comprising purine ribonucleotides andpyrimidine 2′-fluoro-2′-deoxyribonucleotides can be synthesized using aDuraScribe™ T7 Transcription Kit according to the instructions of themanufacturer (EPICENTRE Technologies, Madison, Wis.). Riboprimerscomprising purine ribonucleotides and pyrimidine nucleotides havingother 2′-substituents on the 2′-deoxyribose sugar moiety can besynthesized using a T7 R&DNA™ Polymerase (EPICENTRE Technologies,Madison, Wis.), or a another suitable enzyme, as disclosed in U.S. Pat.No. 5,849,546 and U.S. Patent Application No. 20010049097, bothincorporated herein by reference. Riboprimers containing2′-azido-2′-deoxyribonucleotides and some other modified nucleotides canbe synthesized using the Y639F/H784A double mutant T7 RNA polymeraseenzyme described by Padilla and Sousa (Nucleic Acids Res., 30: e138,2002, incorporated herein by reference).

Strand-Displacing DNA Polymerases of the Invention

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA (“cDNA”) copy from a DNA template. Examples are DNApolymerase I from E. coli and bacteriophage T7 DNA polymerase. All knownDNA-dependent DNA polymerases require a complementary primer to initiatesynthesis. It is known that under suitable conditions a DNA-dependentDNA polymerase may synthesize (i.e., “reverse transcribe”) acomplementary DNA copy from an RNA template, a process that is alsoreferred to as “reverse transcription.”

Some DNA polymerases are able to displace the strand complementary tothe template strand as a new DNA strand is synthesized by thepolymerase. This process is called “strand displacement” and the DNApolymerases that have this activity are referred to herein as“strand-displacing DNA polymerases.” The template for stranddisplacement DNA synthesis using a method of the invention can be alinear or circular ssDNA. If the DNA template is a single-strandedcircle, primed DNA synthesis proceeds around and around the circle, withcontinual displacement of the strand ahead of the replicating strand, aprocess called “rolling circle replication.” Rolling circle replicationresults in synthesis of tandem copies of the circular template. Thesuitability of a DNA polymerase for use in an embodiment of theinvention that comprises strand displacement on linear templates orrolling circle replication can be readily determined by assessing itsability to carry out rolling circle replication. By way of example, butnot of limitation, the ability of a polymerase to carry out rollingcircle replication can be determined by using the polymerase in arolling circle replication assay such as those described by Fire and Xu(Proc. Natl. Acad. Sci. USA, 92: 4641-4645, 1995), incorporated hereinby reference. It is preferred that a DNA polymerase be a stranddisplacing DNA polymerase and lack a 5′-to-3′ exonuclease activity forstrand displacement polymerization reactions using both linear orcircular templates since a 5′-to-3′ exonuclease activity, if present,might result in the destruction of the synthesized strand. It is alsopreferred that DNA polymerases for use in the disclosed stranddisplacement synthesis methods are highly processive. The ability of aDNA polymerase to strand-displace can vary with reaction conditions, inaddition to the particular enzyme used. Strand displacement and DNApolymerase processivity can also be assayed using methods described inKong et al (J. Biol. Chem., 268: 1965-1975, 1993 and references citedtherein, all of which are incorporated herein by reference).

Preferred strand displacing DNA polymerases of the invention are rBstDNA polymerase large fragment (IsoTherm™ DNA polymerase, available fromEPICENTRE Technologies, Madison, Wis., USA), BcaBEST™ DNA polymerase(Takara Shuzo Co., Kyoto, Japan), RepliPHI™ phi29 DNA polymerase (U.S.Pat. Nos. 5,576,204 and 5,001,050 to Blanco et al., incorporated hereinby reference; phi29 is available under the trademark name RepliPHI™ fromEpicentre Technologies, Madison, Wis., USA), SequiTherm™ DNA polymerase(EPICENTRE Technologies, Madison, Wis., USA). Other strand-displacingDNA polymerases which can be used include, but are not limited to, phageM2 DNA polymerase (Matsumoto et al., Gene, 84: 247, 1989), phage ΦPRD1DNA polymerase (Jung et al., Proc. Natl. Acad. Sci. USA, 84: 8287,1987), VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975,1993), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J.Biochem. 45: 623-627, 1974), T5 DNA polymerase (Chatterjee et al., Gene97:13-19, 1991), PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys.Acta, 1219: 267-276, 1994), modified T7 DNA polymerase (Tabor andRichardson, J. Biol. Chem., 262: 15,330-15,333, 1987); Tabor andRichardson, J. Biol. Chem., 264: 6447-6458, 1989); Sequenase™ (U.S.Biochemicals, Cleveland, Ohio, USA), and T4 DNA polymerase holoenzyme(Kaboord and Benkovic, Curr. Biol., 5: 149-157, 1995), all of whichreferences are incorporated herein by reference. IsoTherm™ DNApolymerase (rBst DNA polymerase large fragment; EPICENTRE) is mostpreferred because, in addition to having strand-displacing DNApolymerase activity, it can also be used as a reverse transcriptase forsynthesis of first-strand cDNA from RNA target nucleic acids (e.g., U.S.Pat. No. 6,030,814 of Jendrisak et al., incorporated herein byreference). BcaBEST™ DNA polymerase (Takara Shuzo Co., Kyoto, Japan) canalso be used as a reverse transcriptase as well as a strand-displacingDNA polymerase.

In general, it is desirable that the amount of strand-displacing DNApolymerase in the reaction be as high as possible without inhibiting thereaction. By way of example, but without limitation, IsoTherm™ DNAPolymerase can be used at about 50 units to about 100 units in a50-microliter reaction. Since definitions for units vary for differentDNA polymerases and even for similar DNA polymerases from differentvendors or sources, and also because the activity for each enzyme variesat different temperatures and under different reaction conditions, it isdesirable to optimize the amount of strand-displacing DNA polymerase andreaction conditions for each target sequence and Riboprimer used.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase. It is considered that any DNApolymerase that can perform rolling circle replication in the presenceof a strand displacement factor is suitable for use in embodiments ofthe invention that comprise strand displacement, even if the DNApolymerase does not perform rolling circle replication in the absence ofsuch a factor. Strand displacement factors that permit rolling circlereplication include, but are not limited to, BMRF1 polymerase accessorysubunit (Tsurumi et al., J. Virology, 67: 7648-7653, 1993), adenovirusDNA-binding protein (Zijderveld and van der Vliet, J. Virology, 68:1158-1164, 1994), herpes simplex viral protein ICP8 (Boehmer and Lehman,J. Virology, 67: 711-715, 1993); Skaliter and Lehman, Proc. Natl. Acad.Sci. USA, 91:10,665-10,669, 1994), single-stranded DNA binding proteins(SSB; Rigler and Romano, J. Biol. Chem., 270: 8910-8919, 1995), and calfthymus helicase (Siegel et al., J. Biol. Chem., 267: 13,629-13,635,1992), all of which are incorporated herein by reference.

RNase H Enzymes of the Invention

“Ribonuclease H” or “RNase H” is an enzyme that degrades the RNA portionof an RNA:DNA duplex (or complex). An RNase H can be an endonuclease oran exonuclease. An RNase H enzyme that has endonuclease activity ispreferred for the present invention. The degradation may result inseparation of RNA from an RNA:DNA complex. Alternatively, the RNase Hmay simply cut the RNA at various locations such that portions of theRNA melt off or permit enzymes to unwind portions of the RNA. When usedin an embodiment of the invention, RNaseH enzymes that can be usedinclude, but are not limited to, E. coli RNase H, or Hybridase™Thermostable RNase H (EPICENTRE Technologies, Madison, Wis.), Thermusthermophilus RNase H or Thermus flavus RNase H (U.S. Pat. Nos.5,268,289; 5,459,055; and 5,500,370, incorporated herein by reference).The latter enzymes that are thermostable, and therefore, maintain moreconsistent activity in reactions and are more easily stored and shipped,are preferred in most embodiments of the invention. Other RNase Henzymes that can be used are those that are described by Sagawa et al.,in PCT Patent Publication No. WO 02/16639; and in PCT PatentPublications Nos. WO 00/56877 and AU 00/29742, all of which areincorporated herein by reference. However, in other embodiments it isdesired to use a less thermally stable enzyme, such as E. coli RNase H,because it is easier to inactivate the enzyme in a reaction mixture. Ingeneral, it is preferred that the RNase H enzyme used does not also haveDNA polymerase activity and that the strand-displacing DNA polymeraseused for a strand displacement replication reaction of the presentinvention does not have RNase H activity.

In general, it is desirable that the amount of RNase H in a Riboprimerstrand-displacement replication reaction be as low as possible, so longas the amount of RNase H used liberates the Riboprimer-binding site onthe target sequence so that another Riboprimer can anneal thereto withsufficient efficiency that strand-displacement replication occurs. Theamount of RNAse H that should be used also depends on the length andcomposition of the target-complementary 3′-end portion of the Riboprimerused. As discussed elsewhere herein, sites of cleavage by RNase H varyaccording to the particular sequence. Also, the presence of2′-substituents (such as a 2′-flourine substituent) on sugar moieties ofthe target-complementary portion of the Riboprimer can decrease the ratecleavage by a particular RNase H enzyme. The ratio of strand-displacingDNA polymerase to RNase H enzyme in a strand displacement replicationreaction of the invention also must be optimized. Thus, it is importantthat the reaction comprises sufficient DNA polymerase so that primerextension of the Riboprimer occurs at a faster rate than digestion ofthe target-complementary portion of the Riboprimer that is annealed tothe target sequence. Based on reading the present specification of theinvention, a person with knowledge in the art will understand and knowhow to adjust for different variables in a strand displacementreplication reaction.

By way of example, but without limitation, if IsoTherm™ DNA Polymeraseis used at about 50 units to about 100 units in a 50-microliter stranddisplacement replication reaction, Hybridase™ Thermostable RNase H canbe used in the reaction at about 0.001 to about 0.1 units. Sincedefinitions for units vary for different DNA polymerase and RNase Henzymes and even for similar enzymes from different vendors or sources,and also because the activity for each enzyme varies at differenttemperatures and under different reaction conditions, a person withknowledge in the art will know that it is desirable to optimize theamounts and ratio of strand-displacing DNA polymerase RNase H enzyme andother reaction conditions for each target sequence and Riboprimer used.

Kacian et al., disclosed in U.S. Pat. No. 5,399,491, incorporated hereinby reference, that the number, distribution, and position of putativeRNase H cut sites determine, in part, the usefulness of a given primerand that amplification can be improved by inclusion of intentionalmismatches or insertion of sequences between a transcription promoterand primer in order to affect the number, distribution, and position ofputative RNase H cut sites. Thus, in preferred processes of theinvention for removing RNA from RNA:DNA hybrids following reversetranscription to make first-strand cDNA if an RNA target is used, theRNA target sequence is determined and then analyzed to determine whereRNase H degradation will cause cuts or removal of sections of RNA fromthe duplex upon synthesis of first-strand cDNA. The processes of theinvention include conducting experiments to determine the effect onamplification of the target sequence of the degradation of the RNAtarget sequence by RNase H present in the reverse transcriptase used,including, but not limited to, AMV reverse transcriptase, and both RNaseH-plus and RNase H-minus MMLV reverse transcriptase. The information ofKacian et al., can also be used with respect to designing the optimaltarget-complementary portion of Riboprimers of the invention related tothe RNase H specificities of cut sites for particular enzymes, includingE. coli RNase H or thermostable RNase H enzymes that are stable for morethan 10 minutes at 70° C. (U.S. Pat. Nos. 5,268,289; 5,459,055; and5,500,370, incorporated herein by reference), such as, but not limitedto Hybridase™ thermostable RNase H (EPICENTRE Technologies, Madison,Wis., USA), Tth RNase H, and Tfl RNase H.

With respect to strand displacement replication methods of theinvention, the processes of the invention include conducting experimentsto determine the optimal amount of an RNase H enzyme to use in a stranddisplacement replication reaction mixture on amplification of the targetsequence and degradation of strand-displacement primers comprising RNAor modified RNA (e.g., primers containing 2′-F-dCTP and 2′-F-dUTP thatare made using the DuraScribe™ Transcription Kit). Preferred embodimentsof strand displacement replication reactions will use the minimumconcentration of an RNase H to achieve optimal strand displacement. Athermostable RNase H, such as Hybridase™ RNase H is preferred because itis stable and enzymatic activity is more constant throughout thereaction, making it easier to titrate an optimal level of the enzyme.

Reaction Conditions and Detection Methods of the Invention

Appropriate reaction media and conditions for carrying out the methodsof the present invention are those that permit nucleic acidamplification according to the methods of the present invention. Suchmedia and conditions are known to persons of skill in the art, and aredescribed in various publications, such as U.S. Pat. No. 5,679,512 andPCT Pub. No. WO99/42618, incorporated herein by reference. For example,a buffer can be Tris buffer, although other buffers can also be used aslong as the buffer components are non-inhibitory to enzyme components ofthe methods of the invention. The pH is preferably from about 5 to about11, more preferably from about 6 to about 10, even more preferably fromabout 7 to about 9, and most preferably from about 7.5 to about 8.5. Thereaction medium can also include bivalent metal ions such as Mg²⁺ orMn²⁺, at a final concentration of free ions that is within the range offrom about 0.01 to about 10 mM, and most preferably from about 1 to 6mM. The reaction medium can also include other salts, such as KCl, thatcontribute to the total ionic strength of the medium. For example, therange of a salt such as KCl is preferably from about 0 to about 100 mM,more preferably from about 0 to about 75 mM, and most preferably fromabout 0 to about 50 mM. The reaction medium can further includeadditives that could affect performance of the amplification reactions,but that are not integral to the activity of the enzyme components ofthe methods. Such additives include proteins such as BSA, and non-ionicdetergents such as NP40 or Triton. Reagents, such as DTT, that arecapable of maintaining activities enzyme with sulfhydryl groups can alsobe included. Such reagents are known in the art. Where appropriate, anRNase inhibitor, such as, but not limited to a placental ribonucleaseinhibitor (e.g., RNasin™, Promega Corporation, Madison, Wis., USA) or anantibody RNase inhibitor, that does not inhibit the activity of an RNaseemployed in the method can also be included. Any aspect of the methodsof the present invention can occur at the same or varying temperatures.Preferably, the reactions are performed isothermally, which avoids thecumbersome thermocycling process. The amplification reaction is carriedout at a temperature that permits hybridization of the oligonucleotidesof the present invention to the target sequence and/or first-strand cDNAof a method of the invention and that does not substantially inhibit theactivity of the enzymes employed. The temperature can be in the range ofpreferably about 25° C. to about 85° C., more preferably about 30° C. toabout 75° C., and most preferably about 37° C. to about 70° C. In theseprocesses, the temperature of the transcription steps can be in therange of preferably about 25° C. to about 85° C., more preferably about30° C. to about 75° C., and most preferably about 37° C. to about 55° C.

As disclosed in U.S. Pat. Nos. 6,048,696 and 6,030,814, as well as inGerman Patent No. DE4411588C1, all of which are incorporated herein byreference and made part of the present invention, it is preferred inmany embodiments to use a final concentration of about 0.25 M, about 0.5M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M or between about0.25 M and 2.5 M betaine (trimethylglycine) in DNA polymerase or reversetranscriptase reactions in order to decrease DNA polymerase stops andincrease the specificity of reactions which use a DNA polymerase.

Nucleotide and/or nucleotide analogs, such as deoxyribonucleosidetriphosphates, that can be employed for synthesis of reversetranscription or primer extension products in the methods of theinvention are provided in an amount that is determined to be optimal oruseful for a particular intended use. The oligonucleotide components ofamplification reactions of the invention are generally in excess of thenumber of target nucleic acid sequence to be amplified. They can beprovided at about or at least about any of the following: 10, 10², 10⁴,10⁶, 10⁸, 10¹⁰, 10¹² times the amount of target nucleic acid.Riboprimers and other primers used within the present invention can eachbe provided at about or at least about any of the followingconcentrations: 50 nM, 100 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM, or10,000 nM, but higher or lower concentrations can also be used. By wayof example, but not of limitation, a concentration of one or moreoligonucleotides may be desirable for production of one or more targetnucleic acid sequences that are used in another application or process.The invention is not limited to a particular concentration of anoligonucleotide, so long as the concentration is effective in aparticular method of the invention. In some embodiments, the foregoingcomponents are added simultaneously at the initiation of theamplification process.

In other embodiments, components are added in any order prior to orafter appropriate time points during the amplification process, asrequired and/or permitted by the amplification reaction. Such timepoints can readily be identified by a person of skill in the art. Theenzymes used for nucleic acid amplification according to the methods ofthe present invention are generally added to the reaction mixturefollowing a step for denaturation of a double-stranded target nucleicacid in or from a sample, and/or following hybridization of primersand/or oligos of a reaction to a denatured double-stranded orsingle-stranded target nucleic acid, as determined by their thermalstability and/or other considerations known to the person of skill inthe art.

The amplification reactions can be stopped at various time points, andresumed at a later time. The time points can readily be identified by aperson of skill in the art. Methods for stopping the reactions are knownin the art, including, for example, cooling the reaction mixture to atemperature that inhibits enzyme activity. Methods for resuming thereactions are also known in the art, including, for example, raising thetemperature of the reaction mixture to a temperature that permits enzymeactivity. In some embodiments, one or more of the components of thereactions is replenished prior to, at, or following the resumption ofthe reactions. Alternatively, the reaction can be allowed to proceed(i.e., from start to finish) without interruption.

In some embodiments, the detection of the amplification product isindicative of the presence of the target sequence. Quantitativeanalysis, including analysis in real time, can also be performed in someembodiments. Direct and indirect detection methods (includingquantification) are well known in the art. For example, the amount oftarget sequence in a test sample can be determined by comparing theamount of product amplified from a test sample containing an unknownamount of a polynucleotide having a target sequence to the amplificationproduct of a reference sample that has a known quantity of apolynucleotide with the target sequence. The amplification methods ofthe present invention can also be extended to analysis of sequencealterations and sequencing of the target nucleic acid. The amplifiednucleic acid can be sequenced using any suitable procedure. Many suchprocedures are known. Preferred forms of sequencing for use withamplified sequences produced from some embodiments are nanosequencingmethods described by Jalanko et al., Clinical Chemistry 38: 39-43(1992); Nikiforov et al., Nucleic Acids Research, 22: 4167-4175 (1994);and Kobayashi et al., Molecular and Cellular Probes, 9: 175-182 (1995),and primer extension sequencing, as described in PCT Application WO97/20948, all of which references are included herein by reference.Further, detection could be effected by, for example, examination oftranslation products from RNA amplification products.

Kits and Compositions of the Invention

Important compositions of the invention are Riboprimers. A Riboprimercan be provided for strand-displacement replication of one specifictarget sequence or a Riboprimer can be provided for amplifying amultiplicity of target sequences, such as, but not limited to targetsequences comprising all mRNA targets in a sample. In the latter case, aRiboprimer oligo can be provided that comprises an oligo(dU)_(n)sequence, or an anchored oligo(dU)_(n)X sequence, or a randomizedsequence. Still further multiple specific Riboprimers can be provided inorder to permit amplification of multiple different target sequences inthe same sample.

A kit of the invention can comprise one or more Riboprimers andinstructions for their use in a method of the invention.

A kit of the invention can also comprise all of the enzymes, including astrand-displacing DNA polymerase, such as, but not limited to IsoTherm™DNA Polymerase or RepliPHI™ phi29 DNA Polymerase phi29 DNA Polymerase(U.S. Pat. Nos. 5,576,204 and 5,001,050 to Blanco et al., incorporatedherein by reference; phi29 is available under the trademark nameRepliPHI™ from Epicentre Technologies, Madison, Wis., USA), and aRibonuclease H enzyme, such as but not limited to Hybridase™Thermostable RNase H (all from EPICENTRE Technologies, Madison, Wis.),which are needed for carrying out a strand displacement replication(SDR) method of the invention. The enzymes can be provided in the kitseparately or combined into a single ready-to-use solution containingthe optimal ratio of each enzyme. A kit comprising enzymes forRiboprimer-SDR can also comprise a Riboprimer, or a kit comprisingenzymes for Riboprimer-SDR can be provided without a Riboprimer forcustomers who wish to prepare their own Riboprimers for a specifictarget sequence. Another embodiment of a kit of the invention comprisesa DNA polymerase and a RNase H enzyme for performing Riboprimer SDR, atleast one Riboprimer, and a reaction solution that contains an optimallevel of betaine for performing Riboprimer SDR with the specificRiboprimers in the kit.

A kit of the invention may also optionally comprise additionalcomponents, such as reaction buffers, control substrates, size markers,reagents and instructions to detect the products of a reaction, and thelike, all of which are provided in quantites to match the need for eachcomponent for the number of intended reactions. Still further, a kit mayoptionally contain detailed instructions/protocols and troubleshootingguides.

Example 1 Strand Displacement Replication of ssDNA Template

The template used was a 2 kb concatemeric ssDNA comprising poly-Ubinding sites. 100 picomoles of a mixture of 16 different Riboprimerscomprising 18 ribonucleotides each were obtained. The Riboprimerscomprised U₁₆NN, wherein “N” means A, C, G, or U. In addition, a controlprimer comprising DNA was obtained. Within separate reactions, theRiboprimers and the control primer were annealed to 20 ng of thetemplate DNA by incubating the primers with the template DNA for 30seconds at 95° C. followed by cooling on ice.

A reaction mixture comprising dATP, dCTP, dGTP, and dTTP (each at 0.25mM final concentration), DTT (dithiothreitol, 4 mM final concentration),4 units of RNasin™ Plus (Promega Corporation, Madison, Wis., USA),bovine serum albumin (0.2 mg/ml final concentration), 0.02 units ofRNase H (Hybridase™, Epicentre Technologies, Madison, Wis., USA), 0.2 μgof Single-Stranded DNA Binding Protein (SSB, Epicentre Technologies,Madison, Wis., USA), and 1 μg of phi29 DNA polymerase was added to theprimer-template complexes such that the final volumes were 20microliter. The reactions were incubated at 30° C. for 16 hours,followed by heating at 95° C. for 5 minutes. Aliquots of 4 mircolitereach were analyzed on an ethidium bromide stained 2% TAE agarose gel.

Replication products were observed on the gel (FIG. 3, lane 1),indicating efficient replication. No replication products were observedwhen no template DNA molecule was added (FIG. 3, lane 2) or when thecontrol primer comprising DNA added instead of the Riboprimers (FIG. 3,lane 3).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for amplifying a target nucleic acid sequence comprising atarget nucleic acid: a) hybridizing a Riboprimer to a single strandedDNA template comprising the target nucleic acid sequence, wherein saidRiboprimer comprises: i) only ribonucleotides, or ii) only purineribonucleotides and only pyrimidine nucleotides, wherein at least one ofthe pyrimidine nucleotides is a pyrimidine 2′-deoxyribonucleotide havinga non-canonical substituent, which substituent is neither an H nor anOH, on the 2′-position of the deoxyribose sugar moiety; b) extending theRiboprimer with a DNA polymerase having strand displacement activity; c)cleaving the annealed Riboprimer with an RNase H enzyme such thatanother Riboprimer hybridizes to the template and repeats primerextension by strand displacement, whereby multiple copies of thecomplementary sequence of the target sequence are produced; and d)attaching the multiple copies of the complementary sequence of thetarget sequence onto a solid substrate to make a microarray of theamplified products.
 2. The method of claim 1, wherein a plurality ofRiboprimers is used.
 3. The method of claim 1 wherein the Riboprimercomprises only ribonucleotides.
 4. The method of claim 1 wherein theRiboprimer comprises at least one pyrimidine 2′-deoxyribonucleotidehaving a 2′-substituent on the sugar moiety.
 5. The method of claim 1wherein the Riboprimer comprises at least one pyrimidine2′-fluoro-2′-deoxyribonucleotide.
 6. The method of claim 1 wherein theRiboprimer comprises purine ribonucleotides and pyrimidine2′-fluoro-2′-deoxyribonucleotides.
 7. The method of claim 1 wherein theRiboprimer comprises AMP, GMP, 2′-F-dUMP and 2′-F-dCMP.
 8. A method foramplifying a target nucleic acid sequence comprising a target nucleicacid: a) hybridizing a Riboprimer to a single stranded DNA templatecomprising the target nucleic acid sequence, wherein said Riboprimercomprises: i) only ribonucleotides, or ii) only purine ribonucleotidesand only pyrimidine nucleotides, wherein at least one of the pyrimidinenucleotides is a pyrimidine 2′-deoxyribonucleotide having anon-canonical substituent, which substituent is neither an H nor an OH,on the 2′-position of the deoxyribose sugar moiety; b) extending theRiboprimer with a DNA polymerase having strand displacement activity; c)cleaving the annealed Riboprimer with an RNase H enzyme such thatanother Riboprimer hybridizes to the template and repeats primerextension by strand displacement, whereby multiple copies of thecomplementary sequence of the target sequence are produced; and d)hybridizing the multiple copies of the complementary sequence of thetarget sequence to a microarray of nucleic acid molecules immobilized ona surface of a solid phase.
 9. The method of claim 8, wherein aplurality of Riboprimers is used.
 10. The method of claim 8 wherein theRiboprimer comprises only ribonucleotides.
 11. The method of claim 1wherein the Riboprimer comprises at least one pyrimidine2′-deoxyribonucleotide having a 2′-substituent on the sugar moiety. 12.The method of claim 1 wherein the Riboprimer comprises at least onepyrimidine 2′-fluoro-2′-deoxyribonucleotide.
 13. The method of claim 1wherein the Riboprimer comprises purine ribonucleotides and pyrimidine2′-fluoro-2′-deoxyribonucleotides.